EXCEED THE SPACE PROVIDED. The National Center for Macromolecular Imaging (NCMI) has been supported by NCRR since 1985. We focus on advancing electron cryomicroscopy for structural biology towards atomic resolution. This period, we determined the structures of 7 viruses and molecular machines at 6.5-9.5 A. Most secondary structure elements of the proteins can be seen. This resulted from innovation in instrument set-up, data collection, refinement, feature extraction and visualization. We published 72 manuscripts related to core, collaboration, service, training and dissemination, and sponsored 11 workshops and symposia. In the coming period, we aim 1) to extend the resolution of single particle reconstructions towards 3-4 A; and 2) to transform our experimental and computational steps so that biologists can quickly obtain structures at sub-nanometer resolution. We will install a new cryomicroscope, upgrade instruments, and improve the quality and efficiency of specimen preparation, data collection, pre-processing, refinement, structure analysis, data mining, deposition, visualization, animation and data management. Our staff will partner in some cases with instrument and software developers at other centers and industry. This core development is motivated by 13 collaborative and 16 service projects. These span the spectrum of specimens, including subcellular ordered assemblies, viruses, ion channels, receptors, sensors, molecular machines, multi-subunit enzymes and nano-particles with sizes from -300 kDa to 200 MDa. We will disseminate our technology via publications, workshops, symposia, a book on cryo-EM and video conferencing. We will adopt an open-source code policy for our software. We anticipate a continuous growth in demand for our facility from global users and will emulate the management model of a synchrotron beamline. A committee of prominent scientists will annually advise us. NCMI will lead in integrating all aspects of cryo-EM structure determination to make it competitive for high resolution structural biology of macromolecular assemblies.